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The role of antigen mobility in anti-Rh0(D)-induced agglutination.

Intact human erythrocytes were cross-linked with glutaraldehyde (GA) or dimethyladipimidate (DMA) and tested for their ability to bind [125I]-IgG anti-Rh0(D) and to undergo antibody-mediated hemagglutination. There was no decrease in antibody binding after treatment with GA concentrations up to 1.25% and DMA concentrations up to 1%. Red cells treated with these concentrations of GA and DMA did not agglutinate. The techniques employed to induce agglutination of the cross-linked red cells involved "incomplete" IgG anti-Rho (D) in albumin, "complete" IgM anti-D Rho (D) in saline, and the antiglobulin (Coombs) reaction. The agglutinability of the chemically modified red cells was inversely correlated with the extent of fixation. The dissociation of antibody binding from agglutinability in cross-linked erythrocytes suggests that Rho (D) antigen mobility is required for red cell agglutination. Antigen mobility was manifested by the transition from a relatively monodisperse distribution pattern of Rho (D) antigen sites to one of large aggregates or clusters when agglutination was induced by IgM anti-Rho (D), IgG anti-Rho (D) agglutination of protease modified red cells, and by anti-IgG agglutination of IgG anti-Rho (D)-coated red cells. Antigen clustering was not as prominent in red cells agglutinated by IgG anti-Rho (D) in the presence of albumin. Even though antigen mobility is a prerequisite for antibody-mediated hemagglutination, clustering does not appear to be an absolute requirement. The degree of antigen clustering differs with varying types of agglutination.

Binding Sites, Antibody↗

Lectin-mediated agglutination of amphibian embryonic cells.

Dissociated blastula cells of Xenopus laevis are agglutinated with wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), concanavalin A (Con A) and, to a lesser extent with soya bean agglutinin (SBA). They are not agglutinated with fucose-binding protein. Neuraminidase treatment of cells enhances their agglutinability with RCA and SBA, but has no effect on Con A- and WGA-mediated agglutinability. Treatment of cells with procaine, or xylocaine, has no effect on the cells' agglutinability or on the extrusion of lobopodia. Treatment with colchicine or cytochalasin B either separately or simultaneously has no effect on lectin-mediated agglutinability. Cells treated with cytochalasin B or colchicine and cytochalasin B simultaneously lack lobopodial extensions, while colchicine alone has no effect on these structures. Phenothiazine tranquillizers inhibit agglutination mediated by all of the above mentioned lectins. Lobopodial extensions are absent in cells treated with these compounds. Glutaraldehyde fixation inhibits RCA and WGA mediated agglutinability and reduces the Con A-mediated agglutinability. Results suggest that in this system microtubules and microfilaments are not involved in lectin-mediated agglutination.

Animals↗

Agglutination of Plasmodium falciparum-infected erythrocytes from east and west African isolates by human sera from distant geographic regions.

Plasmodium falciparum-infected erythrocytes (PfE) were collected from acutely infected children in The Gambia and Tanzania and cultured for more than 30 hr until the parasites were mature trophozoites. Sera collected from these countries, other African countries, Asia, and South America were used in the PfE microagglutination test to determine whether PfE from East and West Africa share surface antigens. From the patterns of agglutination reactivity, we identified extensive antigenic diversity in surface antigens, but obtained no evidence for greater differences between isolates from East or West Africa and those within one region. The majority of sera from immune adults from The Gambia, Tanzania, Sudan, Nigeria, or Ghana were pan-agglutinating, and agglutinated all PfE isolates from The Gambia and Tanzania. Some sera from immune adults of Irian Jaya also agglutinated each of the seven African isolates, while others agglutinated many but not all of the isolates, similar to sera from immune adults of Flores, Indonesia. In contrast, sera from nonimmune adults from Colombia agglutinated few of the African isolates. It was remarkable, however, that sera from nonimmune Colombians agglutinated any African isolates. Our results are consistent with the following conclusions: some PfE surface antigen(s) are very diverse; this diversity is a feature of the parasite worldwide; the repertoire of isolate-specific surface antigens, although large, includes antigens that are either identical or antigenically cross-reactive in geographically very distant parasite populations; and African adults have pan-agglutinating antibodies that may contribute to protective immunity. Such pan-agglutinating antibodies could reflect the accumulation of a large repertoire of isolate-specific antibodies. The contribution of antibody against any shared PfE surface antigen to the pan-agglutinating reactivities is unknown and awaits development of the appropriate reagents.

Adolescent↗

Interaction of lectins with surface membrane receptors of animal cells. I. Factors responsible for agglutinability of human, rabbit, and sheep erythrocytes with concanavalin A.

A study of various factors influencing the Con A-mediated cell agglutinability failed to reveal any correlation between the agglutinability and the binding capacity of Con A-agglutinable and non-agglutinable human, rabbit, and sheep erythrocytes. Treatment with trypsin and neuraminidase on the one hand and chondroitin sulphate, polyvinylpyrrolidone and polylysine on the other hand made the agglutination of human erythrocytes possible, promoted the agglutination of rabbit erythrocytes, but had almost no effect on sheep erythrocytes, which agglutinated only after treatment with pronase or polylysine. The negligible effects of low temperature, NaN3, cytochalasine B and theophylline on cell agglutinability indicate that neither membrane fluidity, metabolic energy, cAMP, nor the microfilamentous apparatus are likely to play any important role in the Con A-mediated agglutination of erythrocytes. Differences in agglutinability between erythrocytes obtained from different animal species and subjected to different treatments with enzymes and polymers are explained as being due to alterations in the surface charge and the zeta potential.

Animals↗

Cyclic AMP mediates the concanavalin A agglutinability of mouse fibroblasts.

We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMP(tcs)) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMP(tcs) cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt(2)cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMP(tcs) cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.

Agglutination↗

Agglutination of Pseudomonas aeruginosa by surfactant protein D.

Surfactant protein D (SP-D) is part of the innate host defense system, and may bind and agglutinate invading microorganisms to enhance their removal. The ability of bronchoalveolar lavage (BAL) fluid to agglutinate bacteria and the relationship to its SP-D content are of interest and not yet known. A micromethod on slides was used to assess the agglutination of Pseudomonas aeruginosa by recombinant SP-D and native human BAL fluid. The SP-D-induced agglutination was blocked by calcium depletion, alkaline pH, or the presence of maltose. Twenty-three of 30 BAL fluids from outpatients carrying a chronic tracheostoma clearly agglutinated P. aeruginosa, which was completely inhibited by maltose. The extent of the agglutination correlated weakly to the concentration of SP-D in the BAL fluid, but not to that of SP-A. The functional property, i.e., the agglutination of P. aeruginosa by BAL fluid, was characterized and appeared related in part to the concentration of SP-D. Additional factors, such as the multimeric organization of SP-D, are likely to contribute to the agglutination of microorganisms by BAL or other body fluids. The assay presented will allow the systematic evaluation of small-volume samples for SP-D agglutinating ability from subjects with various lung diseases.

Adolescent↗

Agglutination of Streptococcus mutans serotype C cells but inhibition of Porphyromonas gingivalis autoaggregation by human lactoferrin.

The ability of various forms of human lactoferrin (LF) to agglutinate oral Streptococcus mutans, Strep. sobrinus, Strep. rattus, Strep. sanguis, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans cells was studied spectrophotometrically. Fe3+ saturated LF was unable to agglutinate these bacteria, whereas iron-free LF (apo LF) effectively agglutinated Strep. mutans cells but not the other bacteria. The efficiency and rate of agglutination of Strep. mutans were somewhat lower with apo LF than with human whole saliva. However, secretory IgA, phosphate and whole saliva almost totally abolished the apo LF-mediated agglutination of Strep. mutans, suggesting binding to the same target sites on bacterial cell surfaces, or to each other. The presence of exogenous iron (Fe2+, Fe3+), lactoperoxidase or serum albumin did not affect the agglutination by apo LF. Low Ca2+ (50-100 microns) slightly enhanced the agglutination by apo LF but higher concentrations (0.5-1.0 mM) totally blocked the apo LF-mediated agglutination of Strep. mutans. Both saliva and apo LF significantly delayed the rapid autoaggregation of P. gingivalis cells. Aggregation of P. gingivalis is considered a potential virulence factor and a protective mechanism against the host's cellular defences in the gingival crevice. These findings show a novel, strain-specific antibacterial mechanism for LF against Strep. mutans and P. gingivalis and adds a new compound to the group of agglutinating proteins in human saliva.

Agglutination↗

Saponins can cause the agglutination of phospholipid vesicles.

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4-0.5 by a precipitation assay using 14C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl alpha-L-arabinofuranoside but not by p-nitrophenyl beta-D-glucopyranoside or arabinogalactan. The results indicated that the alpha-L-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.

Agglutination↗

Distribution of leptospirosis among stray dogs in the Okinawa Islands, Japan: comparison of the microcapsule and microscopic agglutination tests.

Three hundred and thirty-eight sera collected from stray dogs in the Okinawa islands were examined for antibodies against Leptospira interrogans using the microscopic agglutination test (MAT) and the one-point microcapsule agglutination test (MCAT). Seventy-eight sera (23%) showed a positive reaction to at least one of the six serovar antigens, and 69 of these reacted with serovar canicola by microcapsule agglutination test. The mixed microcapsule agglutination test detected 68 of the microscopic agglutination test-positive sera, and the 10 remaining were negative by microcapsule agglutination test. On the other hand, a single microcapsule agglutination test which was sensitized with serovar canicola detected 77 of the microscopic agglutination test-positive sera and the remaining one was microcapsule agglutination test-negative.

Agglutination Tests↗

Comparative lectin-binding and agglutination properties of the strain-specific, TA-3-St, and the non-strain-specific, TA3-Ha, murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin.

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific. TA3-Ha line) were compared by binding studies with 125I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl alpha-D-mannopyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Hs cells at a concentration lower than that of TA3-St cells. Epiglycanin, but not asialoepiglycanin, inhibited the agglutination of TA3-Ha cells by WGA.

Agglutination↗

Diagnostic particle agglutination using ultrasound: a new technology to rejuvenate old microbiological methods.

Microbial antigen in clinical specimens can be detected rapidly by commercial test-card latex agglutination, but poor sensitivity is a potential difficulty. Antigen detection by immuno-agglutination of coated latex micro-particles can be enhanced in comparison with the conventional test-card method in both rate and sensitivity by the application of a non-cavitating ultrasonic standing wave. Antibody-coated micro-particles suspended in the acoustic field are subjected to physical forces that promote the formation of agglutinates by increasing particle-particle contact. This report reviews the application of ultrasound to immuno-agglutination testing with several commercial antibody-coated diagnostic micro-particles. This technique is more sensitive than commercial card-based agglutination tests by a factor of up to 500 for fungal cell-wall antigen, 64 for bacterial polysaccharide and 16 for viral antigen (in buffer). The detection sensitivity of meningococcal capsular polysaccharide in patient serum or CSF has been increased to a stage where serotyping by ultrasound-enhanced agglutination is comparable to that achievable with the PCR, but is available more rapidly. Serum antigen concentration as measured by ultrasonic agglutination has prognostic value. Increasing the sensitivity of antigen detection by increasing the acoustic forces that act on suspended particles is considered. Employing turbidimetry to measure agglutination as part of an integrated ultrasonic system would enable the turnover of large numbers of specimens. Ultrasound-enhanced latex agglutination offers a rapid, economical alternative to molecular diagnostic methods and may be useful in situations where microbiological and molecular methods are impracticable.

Agglutination Tests↗

Canine intestinal lactic acid bacteria agglutinated with concanavalin A.

Twenty-six out of 46 representative lactic acid bacteria (LAB) that we isolated from 36 dogs in a previous study were agglutinated by concanavalin A (ConA) at a concentration of 0.1563 mg/ml, while isolates did not agglutinate without the addition of ConA. Amongst the isolates, L. reuteri, L. mucosae, and E. canintestini agglutinated strongly, while L. gallinarum, L. kitasatonis, L. acidophilus, L. saerimneri, B. animalis ssp. animalis, P. acidilactici, and E. hirae did not agglutinate. ConA-agglutination of LAB was specifically inhibited by D-glucose, D-galactose, and D-mannose at a concentration of 1.563 mg/ml. Among the sugars, ConA-agglutination was strongly inhibited by D-mannose, while the inhibition level by D-glucose and D-galactose were lower than that of D-mannose. ConA- agglutination of all the LAB isolates was inhibited by D-mannose, except for L. reuteri (one species) and L. mucosae (two species). ConA-agglutination of Bifidobacterium spp. was inhibited by only D-mannose. Based on our results, ConA-agglutination of LAB seems to be strain-specific, but not species-specific.

Agglutination Tests↗

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa.

AIM: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. METHODS: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. RESULTS: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C. CONCLUSION: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

1-Methyl-3-isobutylxanthine↗

Immune sperm agglutination: are only motile spermatozoa involved?

Human sperm-agglutinating sera was examined using the tray agglutination technique (TAT) with whole diluted semen, and also with a suspension of only progressively motile spermatozoa-obtained from the same semen sample by the penetration of motile spermatozoa in an overlying medium layer. The titres of tail-to-tail (T) agglutinating sera were considerably lower with the latter sperm suspension, but head-to-head (H) agglutinating sera gave similar titres with the two types of sperm suspensions. Attempts to increase titres of T-agglutinating sera with only progressively motile spermatozoa were made by reducing the progression rate of the spermatozoa, and by increasing the concentration of the sperm cells. However, significant titre differences remained. Moreover, the agglutinates obtained were very small, and difficult to observe with the routinely employed magnification of x40. The lower titres still remained where seminal plasma was used as the penetration medium, indicating that a loss of coating antigen(s) was not the cause of lower titres. It seems likely that spermatozoa are attached to each other only weakly in T agglutination, much more weakly than in H agglutination. It is considered that immotile sperm cells that are present in whole diluted semen reinforce the T agglutination process, producing the higher titres.

Antibodies↗

Dextran/glucan binding by Streptococcus mutans: the role of molecular size and binding site in agglutination.

1) S. mutans strains of serotypes a, d and g were strongly agglutinated with soluble glucans and dextran T2000. Homologous glucan did not in all cases produce agglutination. 2) The quantity of low molecular weight dextrans bound (T20 and T70) does not correspond to the agglutination induced by glucan or T2000. 3) The agglutination and binding of high molecular weight glucan by B13 cells was sensitive to heat, trypsin, dextranase, EDTA, SDS and urea, whereas no inhibition of binding of T20 and T70 was seen. 4) Pretreatment of B13 cells with anti-d, or anti-glucan sera, or Con A, RCA I, or RCA II completely inhibited agglutination by T2000 and caused a significant reduction of the binding of glucan. No reduction in the binding of T20 and T70 occurred. 5) An agglutination-negative mutant was agglutinated by sucrose but not by T2000 or high molecular weight glucan. It bound normal levels of T20 and T70. 6) The results indicate that B13 cells possess multiple glucan binding sites and that the site responsible for agglutination consists of both polysaccharide and protein. 7) Inhibition studies on agglutination and adherence using B13 cells indicate that the two processes involve different mechanisms.

Antibodies, Bacterial↗

Lipid-mediated hemagglutination and its relevance to lectin-mediated agglutination.

Oleic acid and dioleoyl phosphatidic acid at low concentrations (20 and 0.5 mu g/ml, respectively) agglutinate rabbit and rat erythrocytes, while dioleoyl phosphatidylcholine is not hemagglutinating up to 0.5 mg/ml. Palmitic acid is not a hemagglutinin and dipalmitoyl phosphatidic acid is a very poor one. A polar lipid fraction obtained from calf thymocytes and a commercial preparation of gangliosides also exhibit pronounced hemagglutinating activity. Modification of the erythrocytes by either trypsin or neuraminidase causes a marked increase in agglutination only with oleic acid, whereas glutaraldehyde fixation of the cells significantly decreases agglutination with oleic acid, dioleoyl phosphatidic acid and calf thymocyte lipids. None of the lipids tested agglutinate freshly drawn human and sheep erythrocytes, but agglutination occurs following fixation of the sheep cells with glutaraldehyde. Lipid-mediated hemagglutination is strongly inhibited by fetuin and bovine submaxillary mucin (0.5 mg/ml). Defatted bovine serum albumin, also at 0.5 mg/ml, inhibits agglutination by oleic acid, whereas agglutination by other lipids is only poorly inhibited if at all. Monosaccharides at concentration up to 0.25 M do not inhibit the hemagglutinating activity of the lipids. Comparison of the hemagglutinating properties of lipids and lectins raises the possibility that the agglutinating activity of crude biological extracts which is not inhibited by mono- or oligosaccharides may be due to lipid constituents. Since agglutination by lipids is species specific, they may serve as mediators in intercellular recognition. The mechanism of lipid-mediated hemagglutination is discussed in terms of current concepts of the fusogenic activity of these compounds.

Animals↗

Effect of cis-dichlorodiammine platinum(II) on the agglutinability of tumor and normal cells with concanavalin A and wheat germ agglutinin.

In this paper we report the effect of cis-dichlorodiammine platinum(II) (cis-platin) on the agglutination of normal and transformed lymphocytes with lectins conconavalin A (Con A) and wheat germ agglutinin (WGA) which is a sensitive test to study the expression of cell surface components. Normal splenocytes showed small degree of cell agglutination with Con A and WGA. This cell agglutination of normal splenocytes with these lectins increases significantly after cis-platin treatment at 37 degrees C. The Dalton's lymphoma cells which show a high degree of cell agglutination with Con A and WGA when treated with cis-platin at 37 degrees C results in decrease in the degree of their cell agglutination with these lectins. Agglutination inhibition studies with alpha-methyl-D-glucopyranoside for Con A and N-acetyl-D-glucosamine for WGA shows that this increase and decrease of cell agglutination after platinum treatment of cells is a specific reaction and not non-specific. Incubation of cis-platin treated normal splenocytes with excess of neuraminic acid results in decrease of cell agglutination with both Con A and WGA. cis-Platin treatment of the cells results in the release of sialic acid from the surface of the cells. A correlation between the release of sialic acid from the cell surface and cell agglutination is also discussed.

Animals↗

Microtiter latex agglutination--quantitative and qualitative equivalent to hemagglutination inhibition for detection of rubella antibody.

Sera were tested for rubella antibody by a standard hemagglutination inhibition method and by latex agglutination in microtiter plates (Virogen Rubella Micro Test). When 157 sera were examined by hemagglutination inhibition and by microtiter latex agglutination for immune status, 149 of the results agreed. The eight discrepant sera (all hemagglutination inhibition-negative, latex agglutination-positive) were tested by a card latex agglutination method (Rubascan) and a passive hemagglutination method (Rubacell). Results, except where noted, agreed with those of the microtiter latex agglutination. One hundred forty-eight of the 149 sera that were in agreement as to immune status were also within a fourfold variation when hemagglutination inhibition and latex agglutination titers were compared. An additional 100 sera were tested for immune status by microtiter latex agglutination and by passive hemagglutination with the single discrepant result agreeing with the latex agglutination when tested by a third method. Both microtiter latex agglutination and hemagglutination inhibition agreed in detection of fourfold or greater rises in each of 20 paired sera.

Antibodies, Viral↗