Ml Of Antiserum Research Articles

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein.

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

Nirantara: A conservative approach to economical use of antisera in short-supply

Background and Objectives: Serological screening for rare antigens may hamper due to scarcity of the reagent antisera. The study aims to develop a conservative approach for scarcely available antisera to maximize outcome from minimum resource. Method: In the LISS tube test for screening the antigen using antiserum, the test supernatant (TS) was harvested after reading the results. The TS was reused in subsequent screening till it showed visible reactivity. The stage at which it ceases to react, the TS was concentrated by carbo-wax (polyethylene glycol) and reused for further screening. Results: Using 8-10 ml of antiserum to rare antigen In(a), we could screen as many as 6868 donors and found 200 donors with In(a+); with 5 ml of anti-Di(a), we could screen 2567 local donors from the Western India; none of them was Di(a+), but four of the 87 Tibetan migrants were positive for the antigen. We could use this approach even on antibody to high-frequency antigen with a slight modification, by adding to the TS the antibody eluted from the sensitized test cell for using in subsequent screening. Conclusion: The recycling of antibody can fruitfully be used several times to conserve the antiserum in short-supply.

PRODUKSI ANTISERUM DAN KAJIAN SEROLOGI CHRYSANTHEMUM B CARLAVIRUS (CVB)

Antiserum production and serological assay of Chrysanthemum B Carlavirus (CVB). Virus identification based on spesific reaction between antigen and antibody in serological assay has been widely applied as a tool for plant virus detection. The aims of this research is to produce antiserum of the CVB by guinea pig immunization using purified CVB of Cianjur isolate. The antiserum was used further for the serological test. Serological methods for detection of CVB were I-ELISA, TBIA, western blot and ISEM. The result showed that guinea pig immunization using 150 µg of purified virus was able to produce 10.75 ml of antiserum. The antiserum produced had high sensitivity for detection of CVB when examined by I-ELISA and TBIA. Besides its low cost, TBIA allows the samples to be blotted on the nitrocellulose membranes in the field and storage of the membranes for later processing in the laboratory. This feature makes it the metode of choice for large-scale CVB surveying.

Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food

Salmonella is one of the most important food-borne pathogens. Early detection of Salmonella is necessary for food safety and hygiene. We have developed an ELISA assay, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant flagellar antigens or H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, α cluster and G complex (McQuiston, J. R. et al., 2004). The genes coding for antigens (fliCgm and fliCi) were cloned into vector pGEX-5X-1 and expressed in the strain E. coli BL21(DE3). The recombinant E. coli was cultured in 200ml LB and the expression of fliCgm and fliCi was induced with 0.4mM IPTG. 2.5mg of each GST-fused protein (GST-H:g,m and GST-H:i) was harvested after purifying by GSTrap FF column. The protein was confirmed by SDS-PAGE analysis and Western Blot with the specific anti-H antigen antibody. 100μg of protein in complete Freund adjuvant was injected to rabbit. Blood collection and booster injection were done every 3 weeks. Terminal blood collection was at the fourth booster injection. 100ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP. The sandwich ELISA procedure utilized the mixture of antibodies. After optimization, the concentration of the mixture of antibodies in the sandwich ELISA method was 250ng of capture antibodies and 100ng of antibody-HRP conjugate. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples was positive of Salmonella presence (OD492>0.3), and only 1% was negative (OD492<0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). In conclusion, the ELISA assay was confirmed the specificity to Salmonella sp. and a very low cross-reactivity to other bacteria presenting in food.

Passive Immunization Approach to Reduce Body Fat in Pigs Using Fat-specific Polyclonal Antiserum

Plasma membrane proteins from pig adipocytes, brain, heart, kidney, liver and spleen were isolated using a 32% sucrose gradient. An adult male sheep was immunized three times at 3-wk intervals with the purified pig adipocyte plasma membrane (APM) proteins. Blood samples were taken from the immunized sheep 12 d after the third immunization. Antiserum showed strong reactivity with APM proteins determined by ELISA, and the reactivity could be detected at dilutions in excess of 1:128,000. Antiserum showed very low binding affinity with proteins isolated from brain, heart, kidney, liver or spleen. Ninety weanling pigs were allocated randomly to three treatment groups and were injected i.p. with 40 ml of antiserum (n = 30) or 20 ml of lyophilized antiserum (21.5 mg/ml; n = 30). A control group (n = 30) received 40 ml of saline, and all pigs were slaughtered at 24 wk of age. The polyclonal antiserum did not change BW or ADG. Carcass percentage of pigs was numerically increased by the antiserum treatment compared with control. Both antiserum treatments did not significantly (p>0.05) affect body composition, including body fat content, relative to the control group.

A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.

Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2

The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ml of antiserum the day before challenge and four pigs used as challenge controls. Pigs were infected by subcutaneous injection with approximately 10 11 colony forming units of S. suis serotype 2. Clinical disease in the pigs that could be attributed to infection with S. suis was reduced from 88 to 35% ( P=0.015). The percentage of pigs with lesions that could be associated with S. suis was reduced from 88 to 22% ( P=0.002) and isolation of S. suis serotype 2 was reduced from five (63%) out of eight pigs in the combined challenge control groups to 3 (13%) out of 23 pigs in the combined treatment groups. These results indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs.

Antibody from hen's eggs against a conserved sequence of the gametophytic self-incompatibility proteins of plants

A convenient way of producing effective antibodies to plant proteins is presented. It takes into account the following facts: (1) many plant proteins are highly glycosylated, thus giving rise to nonspecific antibodies which cross-react with other glycoproteins, (2) it is more common nowadays to know the DNA sequence of a protein coding gene than to have significant amounts of that protein well purified as antigen, and (3) eggs from immunized hens are very convenient sources of large amounts of antibodies. An antibody which specifically detects self-incompatibility proteins (S-proteins) in Solanaceae was isolated. The published cDNA sequences and deduced amino acid sequences of Nicotiana alata (an ornamental tobacco) S-proteins were computer analyzed in order to recognize a peptide with a conserved sequence which would effectively give rise to antibodies. An adequate amount of this peptide was synthesized and part of it was coupled to a macromolecular carrier (bovine serum albumin). Antibodies to this peptide-carrier complex were recovered from egg yolks of immunized hen in amounts corresponding to 300 ml of antiserum per month. From the total immunoglobulin fraction isolated, the antibody specific for S-proteins was affinity purified over 100-fold on a peptide-coupled column. The antibody showed high levels of specificity for putative S-specific sequences in N. alata and in petunia. Also the size, polymorphy, and quantity of the detected antigens were in very good accordance with previously published data. In addition to the present application, this procedure should be useful for a wide variety of proteins.

Passive air-pouch anaphylaxis in rats. I. Induction of anaphylaxis.

Induction of an experimental passive anaphylaxis of the air-pouch type, passive air-pouch anaphylaxis, was carried out in an attempt to induce a reproducible anaphylaxis model suitable for quantitative studies. Rats were injected subcutaneously with 10 ml of air into the dorsal skin to make an air-pouch and with 2 ml of antiserum at an appropriate dilution for passive sensitization, and then 5 ml of air was removed. The challenge with 5 ml of antigen solution into the air-pouch 48 h later provoked mast cell degranulation and increased vascular permeability induced by released histamine. Treatment with monovalent hapten prior to the antigen challenge almost completely inhibited histamine release and plasma exudation to levels similar to those in the nonsensitized group. In this model, mast cell-dependent late-phase allergic reaction, such as leukocyte migration or the increase of plasma exudation following mast cell degeneration, was not observed.

Use of affinity-purified antibodies to measure the in vivo disappearance of antibodies to myotoxin a

M. A. Bober and C. L. Ownby. Use of affinity-purified antibodies to measure the in vivo disappearance of antibodies to myotoxin a. Toxicon 26, 301 – 308, 1988. — Antiserum against myotoxin a was purified using affinity chromatography. Myotoxin a was conjugated to an AffiGel agarose gel bead support and crude antiserum applied to the column. Antibody was eluted with distilled water, acetic acid and phosphate-buffered saline, and the protein concentration in the effluent was estimated by the absorbance at 280 nm. Antibody eluted with distilled water was used to develop an ELISA to detect antibodies to myotoxin in the bloodstream. Mice were injected with either 0.10, 0.15 or 0.20 ml of crude antiserum, and blood samples were taken during a four-week period. Samples were assayed for antimyotoxin using the antibody detection ELISA. Blood levels of antimyotoxin decreased significantly ( P < 0.05) within 1 hr after mice received 0.10 ml of crude antiserum (i.v.). Levels of antimyotoxin in mice given 0.15 and 0.20 ml of antiserum decreased significantly at 3 hr after the injection. Mice given 0.20 ml antiserum had significantly ( P < 0.05) higher amounts of antimyotoxin than mice receiving 0.10 ml antiserum during the 24-hr period after injection. However, after 24 hr all three treatment groups had less than 100 ng antimyotoxin/ml and did not differ significantly from one another. Measurement of antivenom in the bloodstream of snakebite patients might help determine if and when additional antivenom should be administered.

The role of prolactin in lactating rats by passive immunization.

AbstractSome of the physiological roles of prolactin (PRL) in lactating rats were studied by passive immunization. Antibody to rat PRL (A/S PRL) was produced in rabbits immunized with rat PRL. Then the sera of the rabbits were tested for the potency and specificity of antibody to PRL by double antibody radio‐immunoassay technique. One ml of antiserum was inferred to be capable of sufficiently neutralizing endogenous PRL in lactating rats.From the 5th day of postpartum, 11 rats were daily injected intraperitoneally with 1 ml of A/S PRL. In the control group, each of 7 rats was daily injected with 1 ml of normal rabbit serum (NRS). Most pups in the treated group died between the 16th and 20th postpartum days, but all pups in the control group grew normally. Serum oxytocin (OT) levels in the treated group were significantly increased, suggesting that mechanical stimulation of the nipples by pups might increase OT levels, presumably because a lack of PRL decreased milk production. Serum growth hormone (GH) levels in both groups were virtually identical, suggesting that GH could not compensate PRL during the period of passive immunization.

Evidence against a role of prolactin in the timing of ovulation in the turkey.

Two separate experiments in which blood was sampled at 2-h intervals from turkeys hens failed to show a significant change in plasma prolactin (Prl) concentrations in relation to the preovulatory surge of luteinizing hormone (LH) for the first (C1) ovulation of a sequence. Intravenous injection of 125 IU of ovine Prl (NIH-P-S10) or of 1 or 2 ml of antiserum to turkey Prl at varying intervals before C1 ovulation had no effect on the timing or incidence of C1 ovulation. However, injection of Prl before C1 ovulation tended to inhibit ovulation of the second (C2) egg of the sequence, while injection of antiserum to Prl before C1 ovulation tended to either advance or inhibit C2 ovulation. Possibly, the effects of Prl and Prl antiserum on C2 ovulation reflect interference with maturation of the C2 ovarian follicle rather than interference with neuroendocrine processes that regulate the timing of the preovulatory surge of LH. The data for C1 ovulation argue against a change in circulating levels of Prl as a factor in the timing of the preovulatory surge of LH.

Antisera to human renin normalize renin-dependent hypertension in the monkey: therapeutic implications.

Polyclonal antisera raised against pure human renin normalize renin-dependent blood pressure elevation in the monkey (M. fascicularis). In vitro, comparable inhibition of either human or monkey plasma renin by the antisera was demonstrated. In vivo, intravenous infusion of 2 ml of antisera did not change mean arterial pressure of salt-repleted monkeys, however, its administration to salt-depleted monkeys with elevated plasma renin activity lowered mean arterial pressure 10 Torr. A 25 Torr rise in mean arterial pressure and increase in plasma renin activity occurred promptly after inflation of a suprarenal aortic cuff in conscious uninephrectomized monkeys. Administration of 2 ml of antisera to these monkeys normalized mean pressure, which was reduced by an additional 10 Torr if the animals were previously salt-depleted. Maximal hypotension occurred within 1 hour and was sustained for approximately 10 hours. Because of the differing specificities of polyclonal antisera, sera raised in two laboratories against human renin purified from different sources were employed. Identical results were obtained. This is the first demonstration of the use of antisera to inhibit endogenous renin activity in primates and predicts the in vivo efficacy of renin antisera as experimental, diagnostic and therapeutic agents.

Study on role of oxytocin in lactating rats by passive immunization.

SynopsisSome of the physiological roles of oxytocin (OT) in lactating rats were studied by passive immunization. Antibody to synthetic OT (A/S OT) was produced in rabbits immunized with synthetic OT conjugated to bovine serum albumin by the glutaraldehyde method. Then the sera of the rabbits were tested for the potency and specificity of antibody to OT by double antibody radioimmunoassay technique. One ml of antiserum was inferred to be capable of sufficiently neutralizing endogenous OT in lactating rats. This antiserum was proved to inhibit the biological action of OT by Magnus equipment.Fifth day of birth each of 8 rats was daily injected subcutaneously with 1 ml of A/S OT. In the control group each of 6 rats was daily injected with 1 ml of normal rabbit serum (NRS). All pups in the treated group were dead during 11th to 15th day postpartum, though all pups in the control group grew normally. Serum prolactin (PRL) levels in both groups were virtualy identical suggesting that OT might be independent of PRL release. Furthermore the histology of the mammary glands suggested that the role of OT on lactation might not affect the phase of milk production while however affecting milk ejection.

THE HETEROLOGOUS IMMUNE COMPLEX GLOMERULONEPHRITIS:

The pathophysiology, histology and immunohistology of acute and chronic heterologous immune complex glomerulonephritis were investigated in a long-term study in male Wistar rats. The glomerulonephritis showed 3 phases: an initial nephrotic syndrome, a latent phase with stable proteinuria (40 mg/24 h), and a terminal phase with increasing proteinuria and blood pressure, and declining serum protein concentration and creatinine clearance. Antiserum doses of 1.0, 1.5 and 2.0 ml induced maximal proteinuria (112, 257 and 272 mg/24 h respectively) after 14 days whereas normal rabbit serum and 0.5 ml antiserum gave no proteinuria. After 100 days, the rats injected with 1.0 ml of antiserum did not show physiological signs of renal disease; in the rats injected with 1.5 ml of antiserum the disease run a chronic course. Equal amounts of rabbit IgG, rat IgG and rat C3 were found in 10 glomeruli from rats 100 days after injection of 0.5, 1.0 and 2.0 ml (p greater than 0.10). Intramembranous deposits and spike formation were observed in all groups. All changes increased with greater antiserum doses. Chronically diseased animals observed from 500 to 750 days showed deposits of rabbit IgG in the basement membrane, and in most animals small amounts of rat IgG and rat C3 were also observed. This is compatible with a sustained stimulus for antibody formation throughout the course of this type of glomerulonephritis.

Phénotypage Kell et Duffy par une technique de coagglutination sur Groupamatic G 360

By adapting the technique of coagglutination to the Groupamatic, we have developped an efficient rapid method of phenotyping donors for the Kell and Fy a antigens. 340 specimens can be tested per hour; 800 tests can be performed with only 15 ml of antisera. Over 10,000 donor specimens were tested for Kell and 2 470 were simultaneously phenotyped for Fy a. No false negatives were observed. The rate of false positive results, due to autoagglutinins coating the donors' red cells, was the same as that seen with the continuous flow method. We conclude that this technique is suitable for routine use in screening donors for Kell and Fy a antigens.

966 AN ANIMAL MODEL OF LATE PULMONARY RESPONSES TO ANTIGEN CHALLENGE: EFFECTS OF TRANSFUSION WITH “BLOCKING” ANTISERA AND BRONCHODILATOR ADMINISTRATION

Late pulmonary responses (LPR) to mold antigens are a major cause of morbidity in asthmatic patients. We have developed a rabbit model of IgE-dependent LPR to Alternaria aerosol challenge. Rabbits with both IgG and IgE antibodies against Alternaria had blunted LPR measured by increases in total pulmonary resistence (RL) and decreases in dynamic compliance (Cdyn) compared with IgE-only rabbits. To note if passive transfer of IgG would diminish LPR, rabbits were transfused with 30 ml of antiserum heated to remove IgE activity. Marked early pulmonary responses (EPR) (RL=135% of baseline, Cdyn=64%) and LPR (RL 151%, Cdyn=48%) were blunted in three out of four rabbits after transfusion (RL=115%, Cdyn=100% for EPR, RL=119%, Cdyn=100% for LPR). The fourth rabbit had a larger EPR before transfusion compared with the other three (RL=150%, Cdyn =38%), which was partially diminished after (RL=133%, Cdyn=38%). No consistent changes in WBC, differential, platelet count or hemolytic complement were noted in 3 rabbits. Intravenous isoproterenol 6 hrs after challenge did not reverse the LPR. Thus LPR in human asthma as well as in this model is poorly responsive to bronchodilators. Our results suggest that abbrogation of the early, anaphylactic response (e.g. through generation of “blocking” antibody) is important in suppression of the LPR in Alternaria sensitivity. Peripheral leukocyte and complement changes may not be important in the LPR.

The effects of various anaesthetics on rat vasopressin release (author's transl)

Most of the reported studies on the effects of various anaesthetics on vasopressin release have been done based on observations of indirect parameters or bioassay of the hormone, and the results have been quite controversial. The present studies were under-taken, therefore, to evaluate the effect of various anaesthetics on vasopressin release in vivo and in vitro with a radioimmunoassay which we have recently developed.The antibody against arginine vasopressin was raised by immunizing rabbits with lysine vasopressin conjugated with bovine thyroglobulin. The antisera had more than a 90% cross reactivity to arginine vasopressin and DDAVP but virtually no cross reactivity to oxytocin, ACTH, TSH and TRH. Arginine vasopressin was radioiodinated by the chloramine-T method. The radioimmunoassay procedure was as follows : 0.1ml of standard vasopressin or sample, 0.05ml of antiserum (1 : 5,000), 0.1ml of 0.01M phosphate buffer (pH 7.4) and 0.05ml of 125I-AVP (2,500cpm) were incubated for 72 hours at 4°C, and polyethylene glycol was used for the separation of bound and free hormone. The least detectable value in this sytem was 1.5 pg/ml.Ethyl carbamate (1 or 2 g/kg B.W.) or pentobarbital (35 or 60mg/kg B.W.) was injected intraperitoneally into Wistar rats. Ether was administered using an anaesthetic bottle. Blood specimens were obtained from animals by decapitation 20 minutes after the administration of anaesthetics, and vasopressin was extracted from plasma with cold acetone and petroleum-ether successively. After removal of acetone and petroleum-ether under a stream of air, the samples were reconstituted with an assay buffer and served for RIA.The plasma vasopressin level in the anaesthetized rats (except for those which received 1 g/kg B.W. of ethyl carbamate) was 3-7 times higher than that in the control rats which received intraperitoneal saline. The plasma osmolality was also elevated in all experimental groups except those treated with ether and ethyl carbamate (1g/kg B.W.), although there was no correlation between plasma vasopressin level and osmolality.When the neural lobes were examined in vitro following incubation in a Krebs-Ringer bicarbonate buffer containing ethyl carbamate (1 or 2 mg/ml) or pentobarbital (0.05 or 0.1mg/ml), there was no significant change in vasopressin release as compared to control tissues incubated in a plain Krebs-Ringer bicarbonate buffer, although viability of the tissues was proved by a marked increase in hormone release during the final exposure to 60mM KCl.In summary, ether, ethyl carbamate and pentobarbital induced the increase of plasma vasopressin levels in rats in vivo but had no effect on vasopressin release in vitro. These results indicate that these anaesthetics have no direct stimulous effect on the neural lobe, and the elevation of plasma vasopressin levels during anaesthesia is caused through unknown mechanisms such as a change in plasma volume or osmolality, alternation of sensitivity of receptors to those stimuli and possibly a direct stimulation of hypothalamic nuclei. For studies of vasopressin release in rats, ethyl carbamate (1g/kg B.W.) was found to be the most suitable anaesthetic among those presently tested.

Cross-reactivity between immunoglobulins and β2 microglobulin

Cross-reactions between β2 microglobulin (β2m) and human Ig were investigated by the means of two sensitive assays. The first is the capacity of β2m antibodies to lyse human lymphocytes in a complement-dependent lymphocytotoxicity test performed at 15°C. The second is the precipitation of 125I-β2m in the presence of 10% polyethyleneglycol. The latter assay can detect as little as 0.1 ng β2m bound per 1 ml of antiserum. Of 94 anti-human Ig sera prepared in 5 different species, 29 were shown to bind β2m and 13 to be lymphocytotoxic. Both reactions were inhibited by addition of B2m. With 8 sera (4 anti-K, 2 anti-Ig, 1 anti γ 1 + K and 1 anti-Fc IgG) inhibition was also observed after addition of IgG, suggesting that common antigenic determinants were shared by the two molecules, possibly common to β2m and IgK chains. A similar cross-reactivity was demonstrated with 3 out of 14 antisera against β2m. Lymphocytotoxicity and 125Iβ2m precipitation of these 3 sera were partially inhibited by addition of human IgG. Immunoadsorbent experiments with insolubilized β2m or IgG confirmed that the 2 anti-Ig sera studied contained β2m antibodies which could be eluted from IgG immunosorbent, whereas 2β2m antisera contained IgG-binding antibodies which could be eluted from β2m immunosorbent. Homologies of amino acid sequences and similarity in tertiary structure between β2m and Ig domains may account for such cross-reactions. Anti-Ig sera must therefore be absorbed with β2m whenever they are to be used in studies involving lymphocytes.

Inability of passively acquired antibody to protect lambs against experimental pasteurellosis

An experimental model of pneumonic pasteurellosis in sheep was used to investigate the role of serum antibody in resistance to this disease. Lambs which had been vaccinated with a sodium salicylate extract of Pasteurella haemolytica type A1 were protected against challenge with PI3 virus followed by P. haemolytica type A1 7 days later. The majority of untreated lambs and lambs which had received either 200 ml of antiserum to P. haemolytica or 200 ml of control serum intraperitoneally 18 h before infection with P. haemolytica type A1 succumbed to the challenge. Lymphocytes from vaccinated lambs showed a specific proliferative response when exposed to P. haemolytica type A1 sodium salicylate extract, and this response increased after exposure of these animals to P. haemolytica type A1 in aerosol. The results indicate that the humoral immune response alone is incapable of affording protection against experimental pasteurellosis and that cell-mediated immunity may play an important part in resistance to this disease.